Cross-linking collagenous product

ABSTRACT

The present invention relates to a process for cross-linking a proteinaceous material. The process comprises: i) soaking the material to be cross-linked in an agueous solution of high osmolality; ii) incubating the material in an agueous buffer including an amount of a photooxidative catalyst sufficient to catalyze photooxidation of the material; and iii) irradiating the material and the catalyst of step (i) with light that includes a range of wavelengths selectively absorbed by the catalyst. Irradiation is effected under conditions such that cross-linking of the material occurs. In a further embodiment, the present invention relates to a cross-linked product produced by the above-described method.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of copending application Ser. No.07/557,639, filed Jul. 30, 1990, now U.S. Pat. No. 5,147,514, acontinuation-in-part of my co-pending application Ser. No. 07/388,003 ,now abandoned filed Aug. 2 , 1989 and entitled "A Process forCross-Linking Proteinaceous Material and the Product Formed Thereby".

TECHNICAL FIELD

This invention relates , in general, to a process for cross-linking andstabilizing proteinaceous material, and in particular, to a process forphotooxidizing collagenous material in the presence of a photo-catalystto cross-link and stabilize that material. The invention also relates tothe resulting cross-linked product.

BACKGROUND OF THE INVENTION

Reagents and processes currently used for protein cross-linkinggenerally depend upon the incorporation the cross-linking reagent intothe protein matrix to cross-link the ε-amino groups of lysine,hydroxylysine, and/or other groups in the protein. Common cross-linkingreagents in such processes include formaldehyde and glutaraldehyde;other processes include the introduction of a phthaloyl or adipoylmoiety into the protein via phthaloyl dichloride adipoyl dichloride ,respectively, and/or the introduction of a mercaptan for oxidization toa disulfide bond.

The cross-linking processes, reactions and reagents the prior art vary,but most involve incorporating the reagent into or around the protein.For example, recent data by Cheung and Nimni (Connec. Tissue Res. 10:201(1982) and Connec. Tissue Res. 13:109 (1984)) on the cross-linkingreagent glutaraldehyde indicate that when this reagent is used to treatcollagen fibrils, for example, a polymeric-like coating forms around thefibrils, resulting in stiffer collagen matrix.

In contrast, the cross-linking method disclosed and claimed herein doesnot depend upon the incorporation of a cross-linking reagent into thematerial to be cross-linked or the coating of the material with across-linked reagent. The present process involves the use of aphotooxidative dye which acts as a cross-linking oxidation catalyst orpromotor and which can be removed from the cross-linked product.

The use of photooxidative catalysts in various photooxidation processeshas been previously reported (see e.g. , Ray, Method in Enzymol. 11:490(1967); Westhead, Blochem 4:10 (1965); Ray and Koshland, Jr. , J.Biological Chem. 18:409 (1967); and Foote, Science 162:3857 (1968) .However, they do not appear to have been used for cross-linkingproteinaceous materials. For instance, Ray and Koshland, Jr. , supra,used methylene blue and Light to photooxidize the enzymephosphoglucomutase in an attempt to identify the amino acid residues ofthat protein which are essential to the activity of the enzyme byselective destruction of amino acids. Likewise, Westhead, supra,inactivated yeast enolase by photooxidation of histidine residues withthe dye rose bengal.

Excitation of a dye by light has also been used to covalently couple thedye to a protein (Brandt, et al., Biochemistry 13: 4758 (1974)), andthat technique has led to a method of dye-sensitized photolabeling ofproteins (Brantit, et al. , Anal. Blochem. 93:601 (1980) . Although thetechnique is useful for such purposes as the study of the moleculararrangement of proteinaceous membrane components ( Id. ) and proteinconformation (Hemmendorff, et al., Blochem. Biophys. Acta 667: 15(1981)) , the technique does not appear to introduce inter- and/orintra-molecular cross-links into the protein matrix.

A dye-catalyzed process said to be useful for preparing thermostable,irreversibly cross-linked collagenous polymers is described in U.S. Pat.No. 3,152,976. This patent alleges that the product resulting from thatprocess is characterized by certain physical-chemical properties similarto those obtained by prior art tanning processes. However, thesubsequent data presented in that patent do not support a conclusionthat the product of that process possesses the properties of products ofprior art tanning processes which would make that product a usefulbiomaterial for such applications as vascular grafts, heart valves,pericardial patches, injectable collagen, or replacement ligaments ortendoils. Instead, that reference states that the product is moresusceptible to enzymatic degradation than "uncross-linked" collagen.Such results are, of course, totally contrary to the use of such aproduct as, for instance, a heart valve (imagine a heart valve digestedby even tile mildly proteolytic enzyme papain in hours, or even seconds,as described in Example VII of that reference). These seeminglyanomalous results can perhaps in part be explained by the apparentmotivation for making the invention described in that patent, namely theformation of "shaped articles" such as sponges or fibrils (sutures?),ostensibly of a type which can be implanted in the body without typeneed for subsequent removal.

The results reported in the '976 patent can perhaps also be explained bya close examination of the process described therein. For instance, thereference describes the preparation of a "starting material" on whichthe process set out in that patent is conducted by dispersingcollagenous material in aqueous acid solution. Acid has the well-knowneffect of denaturing the protein comprising the collagen fibril. It is,of course, the three-dimensional structure of the proteins comprisingthe collagen fibril which imparts to the fibril the unique properties ofcollagen; change that structure and the protein cannot interact in themanner needed to give rise to those properties. A further explanationfor the results described in that patent is suggested by P. H. vonHipple, "Structural and Stabilization of the Collagen Molecule inSolution" (in Treatise on Collagen, Vol. 1: Chemistry of Collagen, G. N.Ramachandran (Ed.), London: Academic Press Inc. (London) Ltd. (1967),pp. 253-338 at 262), reporting that collagen molecules extracted by acidand neutral salt procedures differ in the extent to which they arecovalently cross-linked, size, shape, interaction properties and rate offiber formation. Although based on preliminary data such that the authorwas careful to point out that results had been reported by otherinvestigators which did not show any differences, subsequentexperimentation supports the existence of such differences.

In light of this prior art, it was surprising to find thatphotooxidation of a protein in the presence of a photo-catalyst andsufficient oxygen, under controlled conditions of pH and temperature,cross-linked and stabilized the collagen to, for instance, enzymaticdegradation, without stiffening the matrix like in conventional tanningprocesses.

OBJECTS OF THE INVENTION

An object of this invention is to provide an effective and efficientmethod for tile non-specific cross-linking of proteinaceous materials.

A further object of this invention is to provide a stable cross-linkedproduct.

A further object of this invention is to provide a collagenous product,and a method of making that product, having physical-chemical propertieswhich make that product suitable for use as a biomaterial for use as anartificial tendon, heart valve, or pericardial patch.

A further object of this invention is to provide a product, and a methodof making that product, which is not antigenic when implanted in amammal.

A further object of this invention is to provide a product, and a methodof making that product, which does not calcify when implanted in amammal.

A further object of this invention is to provide a product, and a methodof making that product, which is not cytotoxic when implanted in amammal and over which endothelial cells are capable of growing.

Other objects of the invention, as well as the several advantages of theinvention, will be apparent to those skilled in the art upon reading thespecification, the examples and the appended claims.

SUMMARY OF INVENTION

In accordance with the present invention, a process is disclosed wherebyproteinaceous material is efficiently and effectively cross-linked andstabilized by subjecting such material to photooxidation in the presenceof a photo-catalyst. In one embodiment, the present invention relates toa process for cross-linking proteinaceous material which comprises: i)soaking the material to be cross-linked in an aqueous buffer of highosmolality; (ii) incubating the material in an aqueous solutionincluding sufficient photooxidative catalyst to catalyze the formationof inter- and intramolecular cross-links by oxidation of the material;and ( iii ) irradiating the material and the catalyst of step (i) withlight that includes a range of wavelengths selectively absorbed by thecatalyst. Irradiation is effected under temperature and pH conditions,and an oxygen concentration, such that cross-linking of the materialoccurs. The present invention also relates to a cross-linkedproteinaceous product produced by the above-described method.

The process of tile present invention provides cross-linked, stabilizedproteinaceous products which are suitable biomaterials for use in tilereplacement and/or repair of diseased or damaged body tissues (medicalprosthetics ) . When so used, the products of the present invention aresuperior to products previously employed, for they retain the mechanicalproperties of the pre-treated material, that is, they remain supple andplaint. In addition, the product is non-immunogenic.

The product of the present invention is further advantageous in medicalprosthetics due to its stability. The cross-linked product resists invivo degradation and calcification when implanted. Therefore thecross-linked product of the present invention is superior to tilebiomaterials known in the art which are susceptible to ordinaryproteolytic degradation and mineralization.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1--Effect of catalyst/irradiation on protein cross-linking andstability as measured by the susceptibility of tissue samples to pepsindigestion.

FIG. 2--Effect of catalyst/irradiation on protein cross-linking andstability as measured by the susceptibility of tissue samples tocollagenase digestion.

FIG. 3--Calcification of implanted cross-linked tissue. Twoglutaraldehyde cross-linked bovine pericardial samples (A and B) areshown (A=X, B=□) , along with glutaraldehyde cross-linked porcineleaflets (0) and a sample of bovine pericardium cross-linked inaccordance with the process of tile present invention (--).

DETAILED DESCRIPTION OF THE INVENTION

The process of the present invention provides an efficient and effectivemethod for cross-linking and stabilizing various proteinaceous materialsincluding, but not limited to, collagen, collagen fibrils and collagenmatrices. The term proteinaceous material as used herein includes bothproteins such as collagen and protein-containing materials such astissues. As a general rule, the particular proteinaceous materialutilized as the starting material is determined by the intended use ofthe product and for that reason, the process of the present inventionhas particular utility for cross-linking collagenous materials. Forinstance, if it is desired to build a heart valve from the product ofthe process of the present invention, the preferred starting material isa material having a high collagen content such as the pericardium, forinstance, bovine pericardium, if the cross-linked product is to be usedas a vascular graft, such starting materials as the aortic arch of ratsor other relatively small animals or the carotid artery of pigs, sheep,or cows are used to advantage. To make injectable collagen, finelyground reconstituted bovine skin collagen is used. The material to becross-linked can also be provided as a tissue sample. Such materials areharvested from the donor animal and immediately immersed in coldbuffered saline for storage, with frequent rinses and/or changes withfresh saline, until processed in accordance with the process describedhereinor solubilized or suspended if finely ground. However, anyproteinaceous material containing tyrosine, tryptophan, and/or histidineresidues is suitable for cross-linking by the present process.

The proteinaceous material to be photooxidized is then immersed,dispersed, or suspended (depending upon its previous processing) in anaqueous media for processing in accordance with the present invention.Suitable media for immersion of the proteinaceous material (for purposesof convenience, the word "immersion" shall be considered to includesuspension and/or solubilization of the proteinaceous material) includeaqueous and organic buffer solutions having a neutral to alkaline pit,preferably a pH of about 6.5 and above because of the denaturationcaused by acid pH. Particularly preferred are buffered aqueous solutionshaving a pH of from about 6.8 to about 8.6. Examples of media that canbe used herein include:

1. water or low ionic strength buffers;

2. phosphate buffered saline;

3. high ionic strength buffers (μ=1.75-3.0); and

4. organic buffers containing potassium or sodium phosphate, orpotassium or sodium chloride, such as a Good's buffer (e.g. , HEPES, TESor BES-Research Organics, Inc.)

The media may also contain tile photocatalyst, which is preferablysoluble therein.

In a particularly preferred embodiment, two media solutions are utilizedfor what is referred to herein as "preconditioning" the collagenousmaterial before irradiation. The material is "preconditioned" in thesense that materials soaked in the first media solution and irradiatedin the second are apparently bettea: cross-linked, e.g., they showimproved mechanical properties and decreased susceptibility toproteolytic degradation. The efficacy of this preconditioning isaffected by the osmolality of tile first media solution, it beingpreferred that solutions of high osmolality be used as the first mediasolution. Particularly preferred are sodium potassium, or organic buffersolutions such as sodium, chloride, sodium phosphate, potassiumchloride, potassium phosphate, and Good's buffers having a pit of fromabout 6.8 to about 8.6, the osmolality of which have been increased byaddition of a solute such as 4M sucrose or other soluble, high molecularweight carbohydrate to between about 393 mosm and about 800 mosm.

The solute added to increase the osmolality of the first media appearsto have an adverse effect on the degree of cross-linking of the productwhen present during irradiation. Consequently, after soaking in thefirst media, collagenous materials are preferably removed therefrom andimmersed in a second media for irradiation. The second media ispreferably an aqueous buffered solution having a pH of from about 6.8 toabout 8.6 in which the photo-catalyst is dissolved. Preferred secondmedia are sodium and potassium phosphate buffers having a pH of fromabout 7.4 to about 8.0 and an osmolality of from about 150 to about 400mosm, 300±10 mosm being particularly preferred.

When the material to be cross-linked is a piece of tissue, tendon, orpericardium, that sample is advantageously immersed sequentially in thefirst media and then in the catalyst-incorporated second media prior tophotooxidation for a total period of time sufficient to allow tissue,dye, and medium to reach equilibrium. When the ratio of theconcentration of the medium to that of the material to be cross-linkedis in the range of from about 10: 1 to 30: 1, equilibrium can generallybe readily achieved. The ratio of the concentrations is generally notcritical, and may be adjusted up or down as desired. Once an equilibriumis reached, the sample is photooxidized in the catalyst-incorporatedmedium. The time required to reach equilibrium varies depending uponsuch factors as, for instance, the temperature of the media solutions,the, osmolality of the first media, and the thickness of the tissue orother sample of proteinaceous material. A period of time as short as afew mixtures or as long as several days may be sufficient, but it hasbeen found that periods of from minutes to hours duration is generallysufficient to allow sufficient time for most collagenous materials andmedia to equilibrate.

Generally speaking, the suitability of a catalyst for use in the presentprocess is dependent upon the ability of the catalyst to be sensitizedinto an exited state (T.) where it serves as a photosensitizer. Thesubstrate then reduces the (T.) state of the sensitizer by electrontransfer. Studies have provided evidence that the substrate reactsinitially with triplet state catalyst, producing secondary reactiveradicals by electron or H atom transfer reactions. See, Spikes andStraight, Ann. Rev. Phys. Chem. 18:409 (1967).

The catalysts contemplated for use herein are photooxidative catalysts(photo-catalysts) that when activated will cause transfer of electronsor hydrogen atoms and thereby oxidize a substrate in the presence ofoxygen. Although varied results are possible depending upon theparticular catalyst utilized, appropriate catalysts include, but are notlimited to, those listed in Oster, et al., J. Am. Chem. Soc. 81: 5095,5096 (1959). Particularly preferred catalysts include methylene blue,methylene green, rose bengal, riboflavin, proflavin, fluorescein, rosin,and pyridoxal-5-phosphate.

The concentration of catalyst in the media will vary based on severalprocess parameters, but should be sufficient to insure adequatepenetration into the material to be cross-linked and to catalyze thephotooxidation of the protein. A typical catalyst concentration rangesfrom about 0.0001%-0.25% (wt/vol); the preferred concentration rangesfrom about 0.01 to about 0.1%.

To achieve maximum cross-linking and stabilization of the proteinaceousproduct, the following steps should be taken: (1) the photooxidativecatalyst should be completely solubilized in the reaction medium priorto use to ensure that the desired dye concentration is achieved; (2) theconcentration of the catalyst in the tissue or suspension should be inequilibrium with that in the surrounding medium; and (3) the catalystsolution should be filtered to remove any sizable particulate matter,including chemical particulates, therefrom.

Because the present process involves primarily an oxidative reaction, toassure completion of the reaction, an adequate supply of oxygen must beprovided during photooxidation. While an oxygen concentration of about20% by volume (referring to the concentration of oxygen in theatmosphere over the media) is preferred to assure sufficient dissolvedoxygen in the media to prevent oxygen content from becoming ratelimiting, concentrations >0% and ranging up to 25% can also be used.Depending upon the temperature at which the proteinaceous material isheld during exposure to light, the oxygen requirement can be met, forinstance, by agitating the solution or otherwise mixing the solution,suspension, or sample during the reaction process. Oxygen concentrationin the atmosphere over the media during irradiation is preferablymaintained in the range of from about 5% to about 20%. Suchconcentrations (again depending upon temperature) can also be achieved,for instance, by bubbling air into the media during irradiation of theproteinaceous material or, if concentrations higher than about 20% aredesired, by bubbling oxygen mixtures or air having an increased oxygencontent into the media.

As with other catalytic or kinetic-type reactions, the temperature atwhich the reaction is run directly affects the reaction rate and theoxygen available in the media. Tests conducted with various mediaranging in pH from about 6.8 up to about 7.4 and having an osmolality of300±10 mosm indicate that as the temperature of the media increases fromabout 4° C. to about 50° C., oxygen concentration drops in roughlylinear fashion from about 11-12 ppm to about 5 ppm. The dye-catalyzedphotooxidation process of the present invention is exothermic, and itis, therefore, preferred that a relatively constant temperature bemaintained during irradiation of the proteinaceous material to preventdenaturation of the proteinaceous material and the driving of the oxygenout of the media by the increase in temperature. Usually, arecirculating bath is sufficient to maintain and control the temperaturewithin the jacketed reaction vessel or chamber but placement of thereaction chamber within a controlled environment such as a refrigeratoror freezer will work as well. As disclosed herein, photooxidationconducted at temperatures ranging from about -2° C. to +40° C. has beenshown to be effective; the preferred temperatures are from about 0° toabout 25° C. To prevent or alleviate denaturation of the proteincomprising the proteinaceous material, temperatures below thedenaturation temperature of that protein are preferred- Likewise,temperatures above the freezing point of the reaction medium are alsopreferred.

It is the combination and/or interaction of the variables oftemperature, pH, and oxygen concentration described herein which isbelieved not to have been previously identified as critical inphotooxidative cross-linking. Hence, the process of the presentinvention is conducted at temperatures low enough to avoid heatdenaturation and pH high enough to avoid acid denaturation of thecollagen or other proteinaceous material during cross-linking. Likewise,temperature is held at a level sufficient to maintain the oxygenconcentration in the media in which the proteinaceous material isimmersed during irradiation.

Once the solution, suspension, or sample is prepared, it isphoto-irradiated, preferably in a controlled system wherein temperature,distance to light source, irradiation energy and wavelength, oxygenconcentration and period of irradiation can be monitored and/ormaintained. The solution, suspension, or sample of proteinaceousmaterial is photo-irradiated under conditions sufficient to causecross-linking. Photooxidation is generally achieved using incandescent,white light or fluorescent light, i.e., visible light, or that portionof light in the visible range that is absorbed by the catalyst.Inexpensive light sources such as household bulbs, fluorescent lightsand flood lamps are suitable for use herein.

The intensity of the light employed, and the length of time required tocross-link a given proteinaceous material will vary depending uponseveral factors. These include: (1) the type and amount of proteinaceousmaterial; (2) the thickness of the tissue sample; (3) the distancebetween the proteinaceous material and the irradiation source; (4) thecatalyst employed; (5) the concentration of catalyst; and (6) the typeand intensity of the light source. For instance, exposure time may varyfrom as little as a few seconds up to as much as about 160 hours. Withregard to the intensity of the light, one or more lights may be used ofintensity preferably ranging up to about 150 watts, preferably held at adistance from about 2.5 cm to 12 cm from the sample surface. Greaterexposure time is required when fluorescent or lower power lights areutilized. These ranges are quite variable; however, they may be easilydetermined for a given material without resort to undue experimentationusing the disclosure and examples provided herein as a guide. In apresently preferred embodiment, the intensity of the light and theexposure time is conveniently expressed in lumen hours, and when commonfluorescent lights are used as the light source, a range of from about100 to about 20,000 lumen hours is utilized for cross-linking mostsamples of proteinaceous material.

Evidence of the cross-linking of proteinaceous material byphotooxidation in the presence of a catalyst in accordance with theprocess of the present invention is provided by several tests. Forinstance, polyacrylamide gel electrophoresis of the irradiated materialin sodium dodecylsulfate (for example, 0.1%) evidences suchcross-linking by a significant decrease in the amount of lower molecularweight material with the simultaneous appearance of high molecularweight material. While amino acid analysis of hydrolyzates ofcross-linked proteinaceous material demonstrates a paucity ofmethionine, tyrosine and histidine (all destroyed by photo-catalyticoxidation), this reduction is not necessarily evidence of cross-linking.For example, if collagen is treated with KI/I₂ solution, derivatizationof tyrosine and histidine occur, essentially eliminating these aminoacids from an amino acid profile without cross-linking, as evidenced bythe lack of change in the gel electrophoretic patterns.

Further evidence of cross-linking is provided by solubility anddigestibility tests such as those set forth in the examples that follow.For instance, cross-linked collagen is generally insoluble such thatsolubility tests provide direct evidence of the degree of cross-linking.The digestibility tests involve incubation of the proteinaceous productwith a proteolytic enzyme such as papain, trypsin, pepsin, or bacterialcollagenase, and the subsequent testing of the media in which theproduct and enzyme are incubated for soluble degradation products oftile cross-linked product. The test is generally accomplished bypelletizing the undigested, cross-linked product and the enzyme bycentrifugation and testing the resulting supernatant for degradationproducts. The latter is particularly useful in light of the destructionof the amino acid histidine by photooxidation; analysis of thesupernatant for histidine content and a comparison of that content tothe amount of an amino acid such as hydroxyproline, which is notdestroyed by photooxidation, in the supernatant provides a particularlysensitive assay for the degree of cross-linking. This comparison can beadvantageously expressed as a ratio of histidine to hydroxyproline(his/hyp ratio), higher his/hyp ratios being indicative of moreeffective cross-linking.

The process disclosed herein is carried out in a batch, intermittent, orcontinuous manner. Following photo-irradiation, the cross-linked productis advantageously subjected to various treatments for the removal of thecatalyst and other chemicals or impurities found therein before beingused as a vascular graft, heart valve leaflet, or other uses listedabove. Multiple rinses in a fresh buffer solution are, for example,used, followed by a least partial de-watering with, for instance,ethanol. The number of rinses and the volume of rinse solution requireddepends upon the mass of the tissue or the suspended material and thecatalyst concentration utilized.

The following non-limiting Examples describe the invention in furtherdetail.

EXAMPLE 1 Pure Collagen Fibrils Cross-linked at 18 C.

Pure reconstituted soluble bovine skin collagen fibrils (0.25 grams)were mixed and suspended in 0,065 liters of 0.02 M sodium phosphatebuffer, pH 7.4, containing 0.01% (wt/vol) methylene green. The collagenfibrils were irradiated in a jacketed water bath maintained at 18° C. Athermometer was placed in the reaction vessel to monitor thetemperature. Two 150 watt floodlights held about 6 cm or 2.4 inches fromthe suspension surface were turned on and the temperature in thereaction vessel began to rise indicating that possibly (1) an exothermicreaction was taking place and/or (2) light energy was being absorbed bythe catalyst in the medium causing an elevation in the temperature ofthe suspension.

The lights remained on and the reaction was allowed to continue for fourhours. The temperature reached an initial maximum of 45° C. which wasthen reduced to, and maintained at about 18° C. for the remainder of thereaction period. The oxygen concentration in the reaction medium washeld at a level sufficient to insure adequate oxygen in the media bykeeping the reaction vessel open to the atmosphere while stirring thereaction mixture vigorously.

The disclosed process was deemed to be effective in cross-linkingproteinaceous material when neither heating of the collagen product to65° C. (which would denature and solubilize the naturally cross-linkednative collagen), nor digestion with pepsin at 15° C. (which would alsosolubilize the native collagen) was successful in breaking it down. Nodenaturation of the collagen product was observed.

EXAMPLE 2 Pure Collagen Fibrils Cross-linked at 0° C.

Following the procedures set forth in Example 1, several samplescontaining approximately 0.25 grams of pure reconstituted bovine skincollagen fibrils were mixed and suspended in 0. 065 liters of 0 . 02 Msodium phosphate buffer, pH 7.4, containing 0.01% (wt/vol) methylenegreen. Photo-irradiation of the suspensions was carried out in jacketedwater baths maintained at a temperature of 0° C. Two 150 wattfloodlights held about 6 cm or 2.4 inches from the surface of eachsuspension were turned on and the reactions were allowed to run forperiods up to 6 hours. The oxygen concentration in the vessels wasmaintained at sufficient levels by keeping the reaction vessels open tothe approximately 20% oxygen concentration of the atmosphere and bystirring (see Example 1).

Photooxidation of the samples was stopped at various time intervalsduring irradiation and pepsin digestion was employed to test thecross-linking and stability of the resulting products. in none of thesamples was any significant degree of solubilization achieved. Evendilute acetic acid at 100° C. or digestion with 5% pepsin (enzyme:substrate) at temperatures up to 18 ° C. did not achieve degradation ofthe cross-linked collagen product.

EXAMPLE 3 Cross-linking of Reconstituted Soluble Collagen Fibrils

Soluble collagen was extracted from 2 year-old bovine skin and purified.Purified reconstituted soluble collagen fibrils were irradiated underthe conditions described in Example 2 in 0.01% and 0 . 1% catalyst for6, 16, 24 , and 48 hours. Attempts were made to dissolve 0.4 mg samplesof the reconstituted soluble collagen fibrils at room temperature afterthey were cross-linked in 0 . 25 nil of 0.5 M acetic acid as well as in0 . 250 ml of 0.1 M TRIS-borate buffer pit 8 . 6 containing 4 M urea and0.2% SDS. The control (non-cross-linked) fibrils dissolved readily at 0°C. and rapidly at room temperature in the latter two solutions. Thecollagen suspensions failed to dissolve when heated to 65° C. Some ofthese suspensions were subjected to PAGE analyses. The reconstitutedsoluble collagen (non-cross-linked) fibrils that dissolved showed normalelectrophoretic patterns. However, when the supernatents of the heatedcross-linked fibrils were run on the gel, the electrophoretic patternswere extremely light and barely visible.

Pepsin and collagenase treatments were used to assess the efficiency ofthe cross-linking reaction. There was apparently very little if anydissolution caused by the pepsin digestion. There was some precipitateleft after collagenase treatment. Aliquots of the supernatents andresidues from each were collected and hydrolyzed. Other aliquots wereremoved for PAGE analysis. The residues from the pepsin digestions wereinsoluble in the TRIS-Borate buffer described above at 65° C.

EXAMPLE 4 Cross-linking Bovine Pericardium Tissue

Using a modified water jacketed reaction vessel similar to that employedin Example 1, a number of samples of bovine pericardium measuringapproximately 2 cm² were irradiated simultaneously with two 150 wattfloodlights held 7 cm to 10 cm or about 2 . 8 to 4 inches from thesamples. Individual samples were removed from the vessel after periodsranging from 2 . 5 to 22 hours of irradiation.

In a first series of experiments, the cross-linking medium consisted of4.0 M sodium chloride with a μ=0.164 sodium phosphate buffer (pit 7.4)containing 0.1% (wt/vol) methylene green. The tissue samples wereequilibrated with the above solution, placed in the apparatus with themedium which was continuously stirred using a magnetic stirrer. Thereaction vessel was then irradiated while temperature of the medium washeld at about 0° C. After various periods of time, the tissue sampleswere removed from the media and decolorized by soaking in μ=0.164 sodiumphosphate buffer (pH 7.4) until the samples were substantially free ofthe catalyst.

To test the effect of the catalyst and the irradiation separately, andthe catalyst/irradiation combination on tissue, control samplescontaining no catalyst or catalyst but no irradiation were run in theabove reaction. After washing for 12 days, changing wash solution threetimes per day, with 0.1 M NH₄ HCO₃, pH 7.9, 0.001 M CaCl₂ solution, thecross-linking and stability of the resulting protein products wereevaluated based on the susceptibility of the tissue samples to pepsindigestion in a 1% pepsin solution in 3% acetic acid at 4 ° C. for 24hours. Reaction of the samples with the enzyme was performed for variedperiods time. The results are depicted in FIG. 1.

Typically, tissue samples irradiated for from 2.5 to 22 hours in tilepresence of the methylene green showed significant decreases in thesolubility of the protein ass compared to the controls. Moreover, a 6hour pepsin digestion of the control tissue yielded approximately 30 nMhydroxyproline (hyp) per mg of tissue, while the same digestion on atissue sample irradiated for 22 hours in the presence of methylene greenyielded values as low as 10 nM per mg. Clearly the disclosed processsuccessfully cross-linked and stabilized the collagen.

EXAMPLE 5 Effect of Reduced Tissue/Volume of Catalyst

Samples of bovine pericardium were soaked in 3.0 M KCl, μ=0.167potassium phosphate buffer and then fewer pieces of pericardium thanutilized in Example 4 were placed in that same buffer including 0.1%methylene green under the same reaction conditions as described inExample 4 and exposed to light for up to 22 hours in these experiments,digestion with pure bacterial collagenase (1% collagenase solution in0.15 M TES buffer, pH 7.5 in 0.001 M CaCl₂ at 37° C. for 6 hours) wasused to evaluate cross-linking and stability. The control sample yielded206 nM hyp per mg of tissue, whereas the sample irradiated for 22 hoursyielded 36 nM of hyp per mg of tissue. These results are depicted inFIG. 2. The reduced tissue susceptibility to collagenase digestiondemonstrated the successful stabilization of the tissue.

EXAMPLE 6 Cross-Linking of Soluble Collagen Fibrils

Soluble collagen was prepared by extraction of two-year old bovinecorium with 1% acetic acid. The soluble collagen was purified by saltprecipitation from the acetic acid solution and two low ionic strengthdialyses from the acetic acid solution, dissolved in 1% acetic acid, andreconstituted into fibrils by dialysis against 0.02 M sodium phosphatebuffer, pH 7.4. Aliquots of the reconstituted collagen were immersed in0.02 M sodium phosphate buffer, pH 7.4 containing 0.01% methylene greenin aluminum-foil covered flasks. The flasks were held at 4° C. in awater bath while bubbling atmospheric air therethrough under two 150 Wflood lamps for 24 hours at a distance of about 3 cm and the foilremoved from a selected number of flasks.

Fibrils from irradiated and non-irradiated aliquots were dialyzedagainst 0.02 M sodium phosphate buffer, pH 7.4 to remove the dye andthen centrifuged and placed in 3% acetic acid. The non-irradiatedfibrils dissolved in the acetic acid while those that were irradiatedremained insoluble, even after heating to 65° C., indicating that theirradiated fibrils had been effectively cross-linked. When thesupernatent from the irradiated fibrils was hydrolyzed for hyp content,none was found, confirming the cross-linking of the collagen.

Fibrils from irradiated and non-irradiated aliquots were also dialyzedfor seven days with three changes per day against the same sodiumphosphate buffer until free of catalyst. A few grams of DOWEX 50 X8resin (20-50 mesh) in the H⁺ form was included with the dialyzing fluidto absorb the catalyst. Samples of each irradiated and non-irradiatedfibrils were then hydrolyzed with 6 N hydrochloric acid for 24 hours at110° C. in vacuo. The hydrolysates were dried and about 50 mg at a timewas subjected to molecular sieve chromatography on a BIO-GEL P2-400 meshcolumn (1.6×100) that had been equilibrated with 0.1 M acetic acid adcalibrated with a 5 mg acid hydrolyzate of NA³ BH₄ -reduced collagenfibrils. The column was monitored by taking small aliquots of thefractions and developed color for amino acids by ninhydrin andscintillation counting of the radioactivity. Void volumes (fractions 33to 48) of natural cross-links of collagen (cross-link fractions) werepooled, lyophilized and subjected to amino acid analysis.

In comparing tile amino acid chromatograms from the irradiated andnon-irradiated fibrils, six distinct new peaks appeared in thehydrolyzate from tile irradiated fibrils between phenylalanine andhydroxylysine. Except for histidine (his), this area is usually a blankarea in a non-irradiated chromatogram, and the presence of these highmolecular weight substances is indicative of the formation ofcross-linked amino acids formed by photooxidation with the dye. Furtherindication was provided by the absence of his in this area, his beingdestroyed by photooxidation.

EXAMPLE 7 Effect of Increased Osmotic Pressure on Cross-Linking

A rectangular illumination cell was constructed from clear plastic withan outer jacket of the same material and tubes communicating with theinner chamber for circulation of media and dye. A frame, comprised ofnarrow strips of plastic including spaced holes therealong for suturingtissue samples thereto, was constructed in a size fitting into the innerchamber of the cell. After suturing a piece of bovine pericardium tothat frame and inserting the frame into the inner chamber, a mediacomprised of 2.8 M potassium chloride, μ=0.164 potassium phosphatebuffer, pH 7.4, including 50% sucrose, was circulated through the innerchamber of the illumination cell. After soaking in the high osmoticpressure media, the tissue was incubated with the media includingmethylene green as described in Example 5, above and illuminated for 24and 48 hours by two 150 watt flood lamps at a distance of about 4.5 cmwhile holding temperature at between -2° C. and 6° C.

After irradiation, small pieces of tissue from each sample were digestedwith pepsin or bacterial collagenase as described in Examples 4 and 5.The following ratios of hyp/mg of tissue in enzyme columns clearlydemonstrate the cross-linking of the tissue samples.

    ______________________________________                                        Time of Irradiation (hrs.)                                                                           Pepsin  Collagenase                                    ______________________________________                                        0       (Control #1)   26      314                                                    (Control #2)   31      314                                            24      (Sample #1)    0       410                                                    (Sample #2)    0       290                                            48                     0       303                                            ______________________________________                                    

Additional tissue samples were further stabilized (without apparentchange in their tactile properties, e.g., tissue texture and suppleness)by reduction of the newly formed iminium bonds by immersion in asolution of NaBH₄ for one hour as demonstrated by the following hyp/mgratios:

    ______________________________________                                        Time of Irradiation (hrs.)                                                                           Pepsin  Collagenase                                    ______________________________________                                        0       (Control #1)   26      314                                                    (Control #2)   7       208                                            24      (Sample #1)    0       180                                                    (Sample #2)    0       170                                            48                     0       170                                            ______________________________________                                    

For purposes of comparison, when a commercially available pericardialpatch prepared by glutaraldehyde tanning was subjected to pepsindigestion at 25° C. and 4° C., ratios of 20 and 16 nM hyp/mg of tissue,respectively, were obtained, and when digested with collagenase at 37°C., 32 and 35 nM hyp/mg tissue ratios were obtained.

EXAMPLE 8 Cross-Linking of Rat Collagen

Soluble BAPN rat type i collagen in 0.5 M HAc was divided into six 4 mlsamples and each sample placed in a dialysis bag with 300 mg NaC1 (nosalt was added to sample 5 and 6). Samples were dialyzed into the highosmotic strength buffer described in Example 7 (samples 5 and 6 weredialyzed into phosphate buffered saline (PBS), p[t 7.4) and 2 ml of 0.2%methylene blue. Samples 2 and 3 were transferred to buffer including0.1% methylene blue in PBS, sample 4 was transferred to PBS including0.01% methylene blue, and samples 5 and 6 remained in PBS. Sample 2 wasexposed to a 150 watt white floodlight located about 7 inches from thesurface of the fluid while holding temperature between about 8 and 12° Cfor eight hours, samples 3 and 4 were exposed for 24 hours under thesame conditions, and samples 5 and 6 were exposed for two hours underthe same conditions. All samples were then dialyzed back into the untilthe solutions were no longer blue and then analyzed by SDS-PAGE asdescribed in Example 3, above. The samples exposed for 24 hours weremore cross-linked than those exposed for eight hours, and all sampleswere more cross-linked than samples 5 and 6.

EXAMPLE 9 Reduced Calcification in Growing Rats

Using the conditions described in Example 3, a series of tissues wereprepared and implanted subcutaneously into the belly of growing threeweek old rats. The control tissues were commercially availableglutaraldehyde cross-linked bovine pericardium (Pericard-A andPericard-B) and porcine leaflets (GL). As can be seen in FIG. 3, thetissue treated according to the invention did not significantly calcifyas compared to the commercial tissue.

EXAMPLE 10 In vivo Biodegration

Bovine pericardium samples treated as described in Example 3 wereimplanted subcutaneously into the belly of adult 6 month old rats. Freshpericardium was used as a control. After three months of implantation,the fresh tissue had resorbed while the treated pericardium remainedintact, again demonstrating the cross-linked nature of the collagenousmaterial.

For purposes of completing this disclosure, all of the references citedhereinabove are hereby incorporated by reference. While the presentinvention has been described in detail for purposes of clarity andunderstanding, it will be clear to one skilled in the art from a readingof the disclosure that changes can be made in form and detail withoutdeparting from the true scope of the invention.

What is claimed is:
 1. The stuble cross-linked collagenous productproduced by the process comprising the steps of:soaking a collagenoustissue sample in an aqueous medium; immersing the soaked sample in anaqueous buffer including a photooxidative catalyst capable of donatingelectrons to the amino acids comprising the collagen fibrils of thecollagenous tissue sample when excited by incident light to form eitherinter- or intramolecular or inter- and intra-molecular cross-links; andirradiating the soaked sample in an aqueous buffer including thecatalyst with light while maintaining the oxygen concentration of theaqueous buffer so as to sensitize the catalyst into an excited statewhich is reduced by oxidative cross-linking of the amino acidscomprising the collagenous tissue sample, the pH being maintained atbetween about 6.8 and about 8.6 and the temperature being maintained atbetween about -2° and about 50° C.
 2. A stable cross-linked productproduced by the method comprising the steps of:soaking collagen fibrilsin a buffered medium having a neutral to alkaline pH which does notdenature the collagen fibrils for a period of time sufficient toequilibrate the concentration of collagen with the concentration of aphotooxidative catalyst solubilized in the medium, and thereafteroxidizing the collagen fibrils to form cross-linkages therebetween byinducing the transfer of electrons from the photooxidative catalyst tothe collagen fibrils in the present of oxygen by exposing the collagenfibrils to light.
 3. The product of claim 2 wherein the medium isbuffered to a pH of about 7.4.
 4. The product of claim 2 wherein theconcentration of catalyst ranges from about 0.0001%-0.25% (wt/vol.). 5.The produce to claim 2 wherein the collagen fibrils are photooxidized ata temperature of from about -2° C. to +40° C.
 6. The product of claim 2wherein the collagen fibrils are exposed to light for about 2.5 to about160 hours.
 7. The product of claim 2 wherein the temperature of themedium is maintained at a selected temperature during the time thecollagen fibrils are exposed to light.
 8. The product of claim 1 whereinthe concentration of catalyst ranges from about 0.001%-0.25% (wt/vol.).9. The product of claim 1 wherein the collagenous sample is exposed tolight for about 2.5 to about 160 hours.